15-hydroxy progesterones



United States atent O IS-HYDROXY PROGESTERONES Josef Fried, NewBrunswick, Richard W. Thoma, Somerville, David Perlman, Princeton, andJohn R. Gerke, Franklin Township, N. J., assignors to Olin MathiesonChemical Corporation, New York, N.Y., a corporation of Virginia NoDrawing. Application May 27, 1955 Serial No. 511,781

15 Claims. (Cl. 260-397.4)

This application is a continuation-in-part of our copending application,Serial Number 372,798, filed August 6, 1953, how Patent No. 2,753,290,issued July 3, 1956.

This invention relates to the synthesis of valuable steroids. 7

One object of this invention is the provision of steroids of theprogesterone series having a 15-hydroxy or 15- acyloxy group, whichcompounds are useful either for their own physiological action or asintermediates in the preparation of physiologically-active derivatives.

The compounds of this invention comprise: (a) 15cchydroxyprogesterone;(b) ISB-hydroxyprogesterone; (c) carboxylic acid esters ofISa-hydroxyprogesterone; and (d) carboxylic acid esters ofISB-hydroxyprogesterone. The preparation of the la-hydroxy and15,8-hydroxyprogesterones of this invention by the microbiologicaloxidation of progesterone is disclosed in said-Patent No. 2,753,290. Thecarboxylic acid esters of l5a-(and 15,9)- hydroxyprogesterone of thisinvention are preferably prepared from the free hydroxy derivatives byreacting the latter with an acylating agent such as a carbonyl halide orcarboxylic acid anhydride, as more fully disclosed hereinafter.

Among the compounds of this invention are those of the general formulawherein R is in either the alpha or beta position and represents hydroxyor an organic carbonyloxy radical. Suitable organic carbonyloxy radicalsinclude the aliphatic carbonyloxy radicals, such as the alkanoyloxyradicals (e.g. lower alkanoyloxy radicals such as acetoxy, propionyloxy,butyryloxy, valeryloxy, caproyloxy, and

, 2,879,280 Patented Mar. 24, 1959 ice Plant Pathology, CornellUniversity, and Phycomyces blakesleeanus (Department of BiologicalSciences, Purdue University) in an aqueous medium containing a source ofnitrogeneous factors and an assimmilable source of carbon and energy, inthe presence of oxygen, and recovering the IS-hydroxyprogesteronesformed. This method is more fully detailed in said Patent No. 2,753,-290 and in the examples following.

The 15-hydroxyprogesterone, thus formed, can then be acylated byreacting the IS-hydroxy steroid with an acylating agent such as acarbonyl halide or "a carboxylic acid anhydride in the presence of abasic agent. Suitable carbonyl halides include the aliphatic carbonylhalides, such as the alkanoyl chlorides (e.g. a lower alkanoyl chloride,such as acetyl chloride, propionyl chloride, butyryl chloride, valerylchloride, caproyl chloride, and enanthoyl chloride), aroyl chlorides(e.g., benzoyl chloride and naphthoyl chloride), cycloalkanoyl chlorides(e.g., hexahydrobenzoyl chloride), aralkanoyl chlorides (e.g., a-toluylchloride and B-phenylpropionyl chloride), and heterocyclic carbonylchlorides (e.g., nicotinoyl chloride, furoyl chloride, and2-thiophenecarbony1 chloride). The preferred carbonyl halides are thehydrocarbon carbonyl chlorides having less than ten carbon atoms, thelower alkanoyl chlorides being particularly preferred. Suitablecarboxylic acid anhydrides include the aliphatic carboxylic acidanhydrides, such as the alkanoic anhydrides (e.g. lower alkanoicanhydrides, such as acetic anhydride, propionic anhydride, butyricanhydride, valeric anhydride, caproic anhydride, and enanthicanhydride), aromatic carboxylic acid anhydrides (e.g. benzoic anhydrideand naphthoic anhydride), cycloalkanoic arrhydrides (e. g.,hexahydrobenzoic anhydride), aralkanoic anhydrides (e.g., a-toluicanhydride and B-phenylacetic anhydride), and heterocyclic carboxylicacid anhydrides (e.g. nicotinic anhydride, 2-furoic anhydride, and2-thiophenecarboxylic anhydride). 'I he preferred'carboxylic acidanhydrides are the hydrocarbon carboxylic acid anhydrides having lessthan nineteencarbon atoms, the lower alkanoic anhydrides beingparticularly preferred. Suitable basic agents are organic bases (e.g.pyridine and collidiue) and inorganic bases (e. g. the alkali salts oflower fatty acids). The acylating reaction is conducted either with anorganic base serving as a solvent, or in an inert organic solvent suchas chloroform, using at least a stoiohiometric amount of acylatingagent.

enanthoyloxy), aroyloxy radicals (eg. aromatic hydrocarbon carbonyloxyradicals, such as benzoyloxy and naphthoyloxy), cycloalkanoyloxyradicals (e.g. hexahydrobenzoyloxy), aralkanoyloxy radicals '(e.g.a-toloyloxy and B-phenylpropionyloxy), and heterocyclic carbonyloxyradicals (e.g. nicotinoyloxy, furoyloxy, and 2-thiophenecarbonyloxy).The preferred organic carbonyloxy radicals, however, are those ofhydrocarbon carboxylic acids having less than ten carbon atoms, thelower alkanoic acid esters being particularly preferred.

To prepare the steroids of this invention, progesterone is subjected tothe action of the enzymes of a microorganism selected from the classconsisting of Penicillium sp. A.T.C.C. 11,598, Streptomyces aureus WC3569 '(Institute of Microbiology, Rutgers University), Strepto- Thecarboxylic acid esters of both the 15u-hydroxyprogesterone andISB-hydroxyprogesterone of this invention are active materials whichpossess progestational activity. Thus, the new steroids of thisinvention can be administered instead of, and in the same manner as,progesterone in the treatment of functional uterine bleeding andamenorrhea. The dosage for such administration is of course dependent onthe relative activity of the particular ester and progesterone. The15u-hydroxyprogesterone and 15 fl-hydroxyprogesterone are alsoutilizable intermediates in the preparation of 14-dehydroprogesterone,as disclosed in application Serial No. 511,783 of Josef Fried, filed oneven date herewith; also in the preparation of 15-ketoprogesterone, asdisclosed in our Patent mycs sp. WC 3676 (Institute ofMicrobiology,-Rutgers University), Colletotrichum antirrhini (Departmentof The following examples are illustrative of the invention (alltemperatures being in centigrade):

EXAMPLE 1 ISa-hydmxyprogestemne (a) FERMENTATION .A medium of thefollowing composition is prepared:

inoculum of Penicillium sp A.T.C.C. 11,598.

CaCO 2.5 Soybean oil 2.2 Progesterone 0.50

Distilled water to make one liter.

The pH of the medium is adjusted to 7.0:01 (with sodium hydroxidesolution); and 100ml. portions of the medium are distributed in 500 ml.Erlenmeyer flasks, and the flasks plugged with cotton and sterilized byautoclaving for 30 minutes at 120. When cool, each of the flasks isinoculated with -10% of a vegetative The flasks are mechanically shakenfor 72 hours in a room maintained'at 25; and the contents of the flasksare pooled, adjusted to pH 4.0:0.2 with sulfuric acid, and filtered bysuction through Seitz filter pads. [The vegetative inoculum used isgrown from stock cultures (lyop'hilized vial or agar slant) for 24-72hours (with or without successive 24-72 hour periods) in a medium of thefollowing composition: 15 g. cornsteep liquor solids; g. brown sugar; 6g. NaNO 0.001 g. ZnS0 1.5 g. anhydrous KH PO 0.5 g- MgSO .7H O; 5 g.CaCO 2 g. lard oil; and distilled water to make one liter, the mediumbeing sterilized by autoclaving for 30 minutes at 120.]

' (b) ISOLATION OF THE IEa-HYDROXYPROGESTER- ONE FORMED 1890 m1. of aculture filtrate obtained as described in section a by fermentation of1.0 g. of progesterone is extracted with four 900 ml. portions ofchloroform. The chloroform solutionsare combined and evaporated todryness in vacuo. The residue, weighing about 358 mg, crystallizesreadily from acetone, and yields a total of about 100 mg. of crystallinematerial melting at 225-30". ilfhis material may be purified bychromatography on a sulfuric acid-washed alumina column. 7 For thispurpose, 38 mg. of the crystalline material is dissolved in 1 ml.chloroform and 3 ml. benzene, and chromatographed on 1 g. alumina;Elution of the column with 200 ml. of a mixture of 1 part of chloroformand three parts of henzene, followed by 80 ml. of a mixture of equalvolumes of chloroform-benzene, yields about 32 mg. of a 150:-hydroxyprogesterone, which after one recrystallization melts at about231-232"; [algal-219- (c, 0.94 in CHC s);,.

kg; 240 m;t(e=16, 500);")\3? 2.93 (OH) 5.92 (sat. CO); 6.02p.(conj. CO);6.19; (conj. double bond).

Analysis.-Calcd. for C21H3Q03: C, 76.33; H, 9.15.

Found: C, 76.14; H, 9.00. Continued elution of the column with 175 ml.chlorofor ields about 9 mg. of material (probably anX,15udihydroxyprogesterone), which after one crystallization melts atabout 251-253; Mi -{4029.

Analysis.Calcd. for C H O C, 72.80; H, 8.73. Found: C, 73.30; H, 9.54.

EXAMPLE 2 1 5 a-h yd roxyp rogesterone (a) 'FERMENTATION A fermentationmedium of the following composition is prepared:

Water to make one liter.

100 m1. portions of the medium are distributed in 500 ml. Erlenmeyerflasks,-and the flasks are pl gg With cotton and sterilized byautoclaving. When c001, each of the flasks is inoculated with 5-10% of avegetative inoculum of Streptomyces aureus WC 3569 which has been grownfor 48-72 hours in a soybean meal-glucose medium. The flasks aremechanically shaken for 72 hours in a room maintained at 25, and thecontents of the flasks are pooled, adjusted to pH 4.0:02 with sulfuricacid, and filtered by suction through Seitz filter pads.

(b ISOLATION OF THE 15a-HYDBOXYPROGESTER- ONE FORMED 2,500 ml. of aculture filtrate obtained as described in section a by fermentation of750 mg. progesterone is treated as described in section b of Example 1,yielding about mg. crude steroids, from which thelSa-hydroxyprogesterone is readily isolated in crystalline form. Theproduct is identical with the 15u-hydroxyprogesterone described insection b of Example 1, both in composition and sterioisomeric form.

EXAMPLE 3 (o) FERMENTATION The same fermentation conditions as describedin section a of Example 2 are employed, except that Streptomyces sp. WC-3676 is employed in place ofthe Streptomyces aureus.

(b) ISOLATION OF THE 15a-HYDROXYPROGESTER- ONE FORMED 2500 ml. of aculture filtrate obtained as described in a by fermentation of 750 mg.progesterone is treated as described in section b of Example 1, yieldingabout 141 mg. of crude product, from which the 15u-hydroxyprogesteronecrystallizes on rubbing with acetone. The product is identical with the15ot-hydroxyprogesterone described in section b of Example 1, asindicated by its melting point, rotation, infrared spectrum, and mixedmelting point.

EXAMPLE 4 ISa-hydroxyprogesterone (a) FERMENTATION The same fermentationconditions as described in section a of Example 1 are employed, exceptthat Colletotrichum antirrhini is employed in place of the Pencilliumsp. A.T.C.C. 11,598.

(b) ISOLATION OF THE 15a-HYDROXYPROGESTER- ONE FORMED 1000 ml. of aculture filtrate obtained as described in a by fermentation of 300 mg.progesterone is treated as described in section b of Example 1, yieldingabout 129 mg. crude product, from which l5u-hydroxyprogesterone may becrystallized.

EXAMPLE 5 ISQ-hydroxyprogesterone (a) FERMENTATION The same fermentationconditions as described in section a of Example 1 are employed, exceptthat Phycomyces blakesleeanus is employed in place of Pencillium sp.A.T.C.C. 11,598.

I (b) ISOLATION OF THE ISB-HYDROXYPROGESTERONE 9 liters of a culturefiltrate obtained as described in a by fermentation of 4.85 g.progesterone is extracted with six 2 liter portions of chloroform. Thecombined chloroform extract is filtered, and evaporated to dryness invacuo. The residue, weighing about 1.31 g., is taken up in 25 ml. 80%aqueous methanol, and the resulting solution extracted with five 25 ml.portions of hexane. The methanol solution is then evaporated to dryness,and the residue (weighing about 1.023 g.) is dissolved with warming in 1ml. of chloroform and 4 ml. of benzene. The resulting solution ischromatographed on 20 g. of sulfuric acid-washed alumina. Elution with400 ml. of a mixture of 1 part of chloroform and 4 parts of benzeneyields 1 Sa-hydroxyprogesterone acetate A solution of 40.3 mg. ofl5a-hydroxyprogesterone (M. P. about 230-232) in 1 ml. of dry pyridineand /1 ml. acetic anhydride is allowed to stand at room temperature for20 hours. After removal of the reagents in high vacuum the residue iscrystallized from acetonehexane. The pure acetate of15a-hydroxyprogesterone has the following properties, m.p. about 175177;[a] +154 (C, 1.00 in chlf.);

13:, 239 m (e =17,700); A221 5.77u(acetyl) 5.89 (ZO-keto), 6.02,u, 620p(A -3-ketone).

Analysis.-Calcd. for C H O (372.49): C, 74.16; H, 8.66. Found: C, 73.66:H, 8.22.

EXAMPLE 7 1513-hydroxyprogesterone-acetare When 15 p-hydroxyprogesteroneis treated with pyridineacetic anhydride at room temperature inaccordance with the procedure of Example 6, or with acetic anhydride insodium acetate at boiling temperature, there is formed a mixturecontaining some starting material and the amorphousISB-hydroxyprogesterone acetate.

EXAMPLE 8 1 Sa-hydroxyprogesterone enan thate Following the procedure ofExample 6, 370 mg. of ISa-hydIoxyprogesterone in 3 m1. of dry pyridineand 0.15 ml. of n-heptanoic anhydride is allowed to stand at roomtemperature for 24 hours. The 15a-hydroxyprogesterone enanthate thusproduced is recovered by the procedure of Example 6.

In a similar manner, 15,3-hydroxypyrogestcrone enanthate can beprepared. Furthermore, by substituting other lower alkanoic anhydrides,such as propiom'c anhydride, butyric anhydride, and valeric anhydride,for the acetic 6 anhydride of Examples 6 or 7 or the heptanoic anhydride.of Example 8, the corresponding propionate, butyrate,

and valerate esters are prepared.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

We claim:

1. A steroid selected from the group consisting of 15-hydroxyprogesterone and hydrocarbon carboxylic acid esters thereof,wherein the acid moiety contains less than ten carbon atoms.

2. ISa-hydroXyprogesterone.

3. ISfi-hydroxyprogesteronc.

4. A hydrocarbon carboxylic acid ester of IS-hydroxyprogesterone,wherein the acid moiety contains less than ten carbon atoms.

5. A lower alkanoic acid ester of IS-hydroxyprogesterone.

6. 15-hydroxyprogesterone acetate.

7. 15a-hydroxyprogesterone acetate.

8. 15 fl-hydroxyprogesterone acetate.

9. ISa-hydroxyprogesterone enanthate.

10. The process for preparing esters of IS-hydroxyprogesterone, whichcomprises reacting, in the presence of a basic agent,15-hydroxyprogesterone with an acylating agent selected from the groupconsisting of a carbonyl halide of a hydrocarbon carboxylic acidcontaining less than ten carbon atoms and a carboxylic acid anhydride ofa hydrocarbon carboxylic acid containing less than ten carbon atoms, andrecovering the ester thus formed.

11. The process of claim 10 wherein the IS-hydroxyprogesterone is15a-hydroxyprogesterone.

12. The process of claim 10 wherein the IS-hydroxyprogesterone is15Bhydroxyprogesterone.

13. The process of claim 10 wherein the acylating agent is a loweralkanoic anhydride.

14. The process of claim 10 wherein the acylating agent is aceticanhydride.

15. The process of claim 10 wherein the acylating agent is heptanoicanhydride.

References Cited in the file of this patent UNITED STATES PATENTS MurrayJuly 8, 1952 Perlman May 31, 1955

1. A STEROID SELECTED FROM THE GROUP CONSISTING OF 15HYDROXYPROGESTERONEAND HYDROCARBON CARBOXYLIC ACID ESTERS THEREOF, WHEREIN THE ACID MOIETYCONTAINS LESS THAN TEN CARBON ATOMS.